A cell-permeable fluorescent prochelator responds to hydrogen peroxide and metal ions by decreasing fluorescence

Described here is the development of two boronic ester-based fluorescent prochelators, FloB (2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-4(5)-[2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene-hydrazinoca rbonyl]-benzoic acid) and FloB-SI (2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-4(5)-[2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)-benzylidene-hydrazinocarbonyl]-benzoic acid) that show a fluorescence response to a variety of transition metal ions only after reaction with H2O2. Both prochelators' boronic ester masks are oxidized by H2O2 to reveal a fluorescein-tagged metal chelator, FloS (4(5)-(2-hydroxy-benzylidene-hydrazinocarbonyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid). Chelation of Fe3+ or Cu2+ elicits a 70% decrease in the emission signal of FloS, while Zn2+, Ni2+, and Co2+ produce a more modest fluorescence decrease. The conversion of FloB to FloS proceeds in organic solvents, but hydrolytic decomposition of its hydrazone backbone is observed in aqueous solution. However, FloB-SI oxidizes cleanly with H2O2 within 1 h in aqueous solutions to produce FloS. Fluorescence microscopy studies in HeLa cells with FloB-SI show that the sensor's fluorescence intensity remains unchanged until incubation with exogenous H2O2, which results in a decreased fluorescent signal. Incubation with a competitive chelator restores the emission response, thus suggesting that FloB-SI can effectively report on a H2O2-induced increase in intracellular labilized metal. (C) 2011 Elsevier B. V. All rights reserved.